Pancreatic acini secrete digestive enzymes and NaCl-rich fluid. both activities are directly regulated by neurohormonal-stimulated Ca2+ oscillations. Therefore, understanding Ca2+ regulation in general and Ca2+ oscillations in particular hold the key to understanding stimulus-secretion coupling in these cells. In the last ten years, we have studied the Ca2+ signal in pancreatic acini using a variety of experimental systems and techniques to follow Ca2+ and Ca2+ fluxes across specific cellular membranes. A central finding of these studies is that Ca2+ itself is the chief regulator of the pathways responsible for Ca2+ regulation. In this respect, our most important recent findings are: a) the agonist-dependent stimulation of the plasma membrane Ca2+ pump, b) the realization that Ca2+ release from internal stores is regulated by a protein kinase phosphatase pair and, c) the possible relationship between the nitric oxide pathway, increased gap junction (GJ) permeability and Ca2+ entry. To build upon and extend these findings, we have developed an agonist antagonist responsive streptolysin O-permeable cell system. In addition, to selectively affect cells within an acinus we puff or inject single cells with solutions of desired composition. We propose to use these techniques to pursue the following: A) Study the Mechanism of Activation of the Plasma membrane Ca2+ Pump by Agonist. This requires an agonist-responsive system that allows free access to the cytosol. Therefore, SLO-permeable cells and membranes isolated from them will be used to evaluate the role of phospholipase C activation, Ca2+ calmodulin and the cytoskeleton architecture in agonist-mediated pump activation; B) Probe the Mechanism of Regulation of Ca2+ Release by Ca2+. The biphasic regulation of Ca2+ release by Ca2+ involves the sequential activation of Ca2+ -dependent protein kinase and phosphatase. Agonist stimulation profoundly affects this regulation. To unequivocally identify the kinase phosphatase pair, we will use the SLO-permeable system to activate the Ca2+ release channel by IP3 or agonist and inactivate the channel by ant-agonist while modulating the activity of specific protein kinases and phosphatases, C) Establish a Relationship Between Ca2+ Entry, Nitric Oxide and Gap Junctions. Our recent measurement of GJ permeability with FRAP and the patterns of Ca2+ oscillations evoked by focal and global stimulation suggest a relationship between these activities. We propose to use FRAP and imaging of Ca2+ to study the role of the NO pathway in regulating Ca2+ entry and GJ permeability; D) Study Regulation of Ca2+ mechanisms discovered in pursuing aims a-c in the overall signal of Ca2+ oscillations stimulated by agonists or an agonist-independent manner will be studied by injection of specific modulatory proteins and compounds. We hope that the proposed studies will uncover novel aspects of Ca2+ regulating and lead to better understanding of Ca2+ signaling and their role in the physiological function of pancreatic acini.